This invention is in the area of immunology/biochemistry, and particularly concerns the development and production of compositions and methods for identifying inhibitors of protein hormone release, and prophylactic and therapeutic uses of the inhibitors for treating diseases associated with elevated levels of the hormones. More specifically, the invention facilitates the identification of compositions and methods for identifying inhibitors of a TNFxcex1 convertase. These inhibitors may be used to treat a variety of diseases, particularly sepsis, rheumatoid arthritis, cachexia, AIDS and autoimmune diseases, and thus affords the physician alternate treatment regimes.
In the United States alone nosocomial bacteremia develops in about 194,000 patients per year, and of these about 75,000 die. Maid, D. G., 1981, Nosocomial Infect., (Dikson, R. E., Ed.), page 183, Yrke Medical Books, U.S.A. Most of these deaths are attributable to six major gram-negative bacillixe2x80x94Pseudomonas aeruginosa, Escherichia coli, Proteus, Klebsiella, Enterobacter and Serratia. The current treatment for bacteremia is the administration of antibiotics which have limited effectiveness in treatment of septic shock frequently associated with bacterial infection.
The precise pathology of the septic shock sometimes associated with bacteremia is not completely elucidated. Nevertheless, it is known that certain bacterial endotoxins called lipopolysaccharides (LPS), are the primary causative agents. LPS consists of at least three significant antigenic regions: lipid A; core polysaccharide; and O-specific polysaccharide. The latter is also referred to as O-specific chain or simply O-antigen. The O-specific chain is a long-chain polysaccharide built up from repeating polysaccharide units. The number of polysaccharide units differs among different bacterial species and may vary from one to as many as six or seven monosaccharide units. While the O-specific chain varies among different gram-negative bacteria, the lipid A and core polysaccharides are similar if not identical.
LPS initiates a cascade of biochemical events that can lead to the death of the patient. It is widely believed that an early result of exposure to LPS is the stimulation of macrophage cells and the production of tumor necrosis factor alpha (TNFxcex1). Based on this belief, considerable effort has been expended to produce neutralizing antibodies and other molecules that could inhibit the effects of TNFxcex1 and which could serve as valuable clinical adjuncts to the standard antibiotic therapies used in the treatment of septic shock. Tracey et al., Nature 330:662 (1987).
While many cell types are capable of expressing TNFxcex1, including for example, T and B lymphocytes, fibroblasts, and endothelial cells, the principal source is macrophages. TNFxcex1 has been reported to exist in both membrane-bound and soluble secreted forms. Decker et al., J. of Immunol. 138:957 (1987); Kriegler et al., Cell 53:45 (1988). Human TNFxcex1 has been cloned and consists of a 17 kD polypeptide, plus a 76-amino-acid pro sequence containing the residues that appear to be responsible for anchoring proTNFxcex1 as a type II membrane protein. The 17 kD molecule is a key agent involved in initiating the biochemical cascade responsible for sepsis. TNFxcex1 may exist as both a membrane-bound 26 kD form, and a soluble form corresponding to the 17 kD species. Kreigler et al., Cell 53:45 (1988). The 26 kD form is the precursor, or prohormone, of the mature 17 kD molecule. The two forms of TNFxcex1 may have different biological effects, primarily as a result of differences in tissue distribution. Kriegler et al. supra
Because TNFxcex1 plays a key role in the sequelae of sepsis and is believed to be an inflammatory agent in diseases, there is a need to identify and develop anti-TNFxcex1 prophylactics/therapeutics. Anti-TNFxcex1 antibody has shown promise in in studies employing a baboon model system. (Hinshaw et al., Circulatory Shock 30:279 (1989)). However, these studies involve non-human anti-TNFxcex1 and non-human anti-TNFxcex1 antibody. In addition, TNFxcex1 is involved in inducing the expression of human immunodeficiency virus (HIV) in human cells that carry latent virus. Folks et al., PNAS (USA) 86:2365 (1989).
TNFxcex1 also plays a role in various autoimmune diseases, particularly rheumatoid arthritis. Duff et al., International Conference on Tumor Necrosis Factor and Related Cytotoxins, 175:10 (1987). Compounds or methods for inhibiting TNFxcex1 action will have considerable application for the treatment of a variety of diseases of immunologic origin. As described in Fiers et al., FEBS Lett. 285:199 (1991), a variety of other serious human conditions including cerebral malaria, graft-versus-host disease and ischemia/reprefusion injury, are also associated with TNFxcex1 biological activity.
In addition to anti-TNFxcex1 antibodies, other molecules with TNFxcex1 inhibitory activity are being sought. Non-antibody TNFxcex1 inhibitors are described by Seckinger et al., Exp. H. Med. 167:151 (1988), and Seckinger et al., Biol. Chem. 264:11966 (1989), and in European Patent Application No. 88830365.8, inventors Wallach et al. The inhibitors are present in the urine of febrile patients, and are reported to have molecular weights of about 27,000-33,000. These inhibitors are reported to be soluble forms of the TNFxcex1 receptor. Although these molecules exhibit TNFxcex1-inhibitory activity, neither of the inhibitors is shown to be effective in the treatment of septic shock in humans.
From the foregoing discussion it is apparent that there is a need to identify and develop additional modulators of TNFxcex1 activity, both antibody-based or otherwise, that are efficacious in the treatment of TNFxcex1-mediated diseases.
An approach to modulating the activities of TNFxcex1 according to the present invention involves the inhibition of TNFxcex1 convertases, for example PR-3, which are capable of converting locally produced, membrane-bound proTNFxcex1 to TNFxcex1 which contributes significantly to the pathologic processes of diseases such as septic shock and such as preventing or inhibiting the formation of the 17 kD, or lower molecular weight forms of TNFxcex1 might be a valuable prophylactic for the prevention of AIDS in HIV-positive patients by preventing the expression of virus that is latent in the patient and others described above.
In its most general form, the invention described herein presents methods and compositions for inhibiting the production of a mature form of TNFxcex1, from its prohormone precursor, proTNFxcex1 in its 26 kD form or multimers thereof and its soluble 20 kD form or multimers thereof. These compositions are useful for preventing or treating diseases in patients associated with elevated levels of mature TNFxcex1 including septic shock, AIDS, cerebral malaria, graft versus host disease, ischemia/reperfusion injury, rheumatoid arthritis, and cachexia. The invention also relates to methods (e.g. colorimetric and autoradiographic) for identifying molecules that inhibit the production of a mature form of TNFxcex1. Such inhibitors are distinguishable from anti-TNFxcex1 antibody or soluble TNFxcex1 receptor, which block TNFxcex1 activity by binding to TNFxcex1.
This method may be used to identify medicaments such as prophylactics and/or therapeutics for the treatment of diseases associated with the production of mature TNFxcex1 such as those discussed above. Medicaments identified by this method interfere with the cleavage of the 26 kD proTNFxcex1 prohormone by enzymes termed convertases. Thus, these medicaments inhibit the production of lower molecular weight molecules (i.e., circulating mature forms of TNFxcex1 having subunits of 17 kD molecular weight) which play a role in the induction of xe2x80x9cseptic shockxe2x80x9d associated with sepsis and other diseases. Specifically, preferred inhibitors as described herein interfere with the activity of a TNFxcex1 convertase to prevent removal of the N-terminal portion of the 26 kD molecule including at least the 76 amino-acid signal sequence to produce a mature form of TNFxcex1 such as the 17 kD TNFxcex1. The invention also includes a class of compounds that are both inhibitors of a TNFxcex1 convertase and that are effective in the prevention and/or treatment of septic shock. Compounds in this class include, for example, anti-convertase antibody, muteins of the prohormone form, and proteins or peptides that compete with the 26 kD form of TNFxcex1 for binding to the convertase. Also part of the invention are small molecular weight compounds that specifically inhibit a class of proteases that includes TNFxcex1 convertases, or preferably, show selective specificity for inhibition of TNFxcex1 convertase. Such small molecular weight compounds are exemplified by, but are not limited to compounds such as the peptide diphenyl phosphonates Boc-X-p(OPh)2, wherein X is a peptide selected from the group consisting of Val-Pro-Val, Ala-Pro-Val, and Val-Pro-His.
Additionally, the present invention is directed to a TNFxcex1 convertase purified to near homogeneity, the amino acid sequence of said convertase, and methods for expressing a recombinant form of TNFxcex1 convertase. One purified human TNFxcex1 convertase contains an N-terminal amino acid sequence essentially identical to human PR-3, a known neutrophil protease having the same molecular weight. The present invention is also directed to various inhibitors of TNFxcex1 convertase and methods for detecting inhibitors.
More specifically, the present invention is directed to small molecules that specifically inhibit TNFxcex1 convertases.
The invention is also directed to a method for treating diseases such as septic shock, cerebral malaria, rheumatoid arthritis, AIDS, cachexia, ischemia/reperfusion injury, and graft-versus-host disease by administering a convertase inhibitor such as a PR-3 inhibitor. Pharmaceutical compositions and medicaments comprising the convertase inhibitors of the present invention represent still another aspect of the present invention.
In one aspect of this invention, a method is provided for identifying a substance for prophylactic or therapeutic treatment of a disease caused by, exacerbated by, or associated with tumor necrosis factor (TNFxcex1) produced from a proTNFxcex1 by cleavage of said proTNFxcex1 by a TNFxcex1 convertase, the method comprising the steps of: (a) contacting the proTNFxcex1 with an amount of the TNFxcex1 convertase effective for cleaving the proTNFxcex1; (b) measuring the conversion of the proTNFxcex1 to the mature TNFxcex1 in step (a); (c) repeating steps (a) and (b) further including a molecule sought to be identified as a substance for prophylactic or therapeutic treatment of diseases caused by, exacerbated by, or associated with the soluble TNFxcex1; (d) measuring the conversion of the proTNFxcex1 to the mature TNFxcex1 in step (c); and (e) comparing the conversion measured in step (b) with the conversion measured in step (c) to determine whether the molecule is a suitable prophylactic or therapeutic of diseases caused by mature TNFxcex1. The measuring steps include but are not limited to colorimetric methods and autoradiographic methods. Possible source of such inhibitors are libraries of known elastase inhibitors.
In yet another aspect of the invention, a method is provided for treating a patient having a disease or susceptible to a disease caused by, exacerbated by, or associated with a mature TNFxcex1 produced from a proTNFxcex1 by cleavage of said proTNFxcex1 by a TNFxcex1 convertase, the method comprising administering to a patient in need of such treatment an effective amount of an inhibitor of a TNFxcex1 convertase. In a preferred embodiment, the disease is selected from the group consisting of sepsis, rheumatoid arthritis, cachexia, cerebral malaria, AIDS, autoimmune disease, and graft-versus-host disease.
In a further aspect of this invention, a pharmaceutical composition is provided for the treatment of a disease caused by a mature TNFxcex1 produced from a proTNFxcex1 by cleavage of said proTNFxcex1 by a TNFxcex1 convertase, the composition comprising an effective amount of an inhibitor of a TNFxcex1 convertase and a pharmaceutically acceptable carrier or excipient.
In another of its aspects, the present invention provides purified and isolated polypeptides and variants thereof which display biological/biochemical properties specific to the proenzyme and mature active forms of human neutrophil protease PR-3. Biological/biochemical properties of preproPR-3 (SEQ ID NO: 23) include a structural amino acid sequence of the mature, active PR-3 (SEQ ID NO: 27) enzyme in addition to a dipeptide, characteristic of the zymogen form of the enzyme, between the leader and amino acid sequences of mature, active PR-3.
Yet another aspect of the present invention is a purified polypeptide comprising the pro-form of a granzyme having an N-terminal amino acid sequence of X-E, wherein X represents a variable amino acid and wherein E represents glutamic acid and wherein said N-terminal sequence renders the granzyme catalytically inactive and wherein the granzyme when active catalyzes the cleavage of a membrane bound protein hormone or receptor ligand to generate the free, soluble form of the ligand.
According to another aspect of the invention, isolated polynucleotides (e.g. DNA and RNA transcripts thereof) encoding preproPR-3 and variants thereof which display useful properties of preproPR-3 are provided. Preferred DNAs of the invention include genomic and cDNA as well as wholly or partially chemically synthesized DNA. The most preferred polynucleotide sequence is set forth in SEQ ID NO: 22. Replicas (i.e., copies of isolated DNA sequences made in vivo or in vitro) of the invention are contemplated. Also provided are autonomously replicating recombinant constructions such as plasmid and viral DNA vectors incorporating preproPR-3 sequences and especially vectors wherein DNA encoding preproPR-3 or a preproPR-3 variant is operatively linked to an endogenous or exogenous expression control DNA sequence.
According to another aspect of the invention, host cells, preferably eukaryotic cells, are stably transformed with DNA of the present invention in a manner allowing the desired polypeptide to be expressed and therein processed and secreted. Trichoplusia ni (Tn5) insect cells infected with a viral vector containing PR-3 sequences of the present invention are also preferred. Spodoptera frugiperda insect cells (Sf9) infected with a viral construct containing a polynucleotide encoding preproPR-3 are variants thereof are most preferred. Other host cells contemplated by the present invention include mammalian cells such as CHO cells or human 293 cells.
Another aspect of the present invention is directed to a process for the large-scale production of the pro-form of granzymes such as proPR-3 (SEQ ID NO: 25) and variants thereof, wherein the host cells of the present invention are grown in a suitable culture medium and desired polypeptides are isolated from the cells or from the medium in which the cells are grown. The pro-form of the granzymes produced by these methods may be activated by the enzymatic removal of the N-terminal amino acid sequence using enzymes such as dipeptidyl peptidases and more particularly, dipeptidyl peptidase I.
ProPR-3 and novel PR-3 variants thereof have not been obtained from natural cell sources. However, the present invention for the production of active recombinant PR-3 (SEQ ID NO: 27) or PR-3 variants is valuable for identification of TNFxcex1 convertase inhibitors. The present invention also provides for the production of useful amounts of recombinant PR-3 (SEQ ID NO: 27) or PR-3 muteins in a non-pyogenic form suitable for clinical use in humans. Another aspect of the present invention are PR-3 muteins in which glycosylation has been blocked by substitution at N-linked glycosylation sites thereby improving the homogeneity and possibly the crystallizability of recombinant PR-3.
Also comprehended by the present invention are antibody substances (e.g., monoclonal and polyclonal antibodies, single-chain antibodies, chimeric antibodies, CDR-grafted antibodies and the like) which are specific for proPR-3 (SEQ ID NO: 27), PR-3, and PR-3(SEQ ID NO: 27), variants and muteins. Antibody substances can be developed using isolated natural or recombinant proPR-3, PR-3, PR-3 variants or muteins thereof. Most preferred are antibody substances which are specific for the amino acid sequences including those residues unique to the pro-form, e.g. antibodies specific for the conformation of the inactive proPR-3.
In another of its aspects, the invention is directed to the treatment of cases of undesirable B cell/T cell interactionss comprising treating T cells with a therapeutic amount of PR-3 effective to release membrane bound cytokines which mediate B cell/T cell interactions.